Chemokine receptor 88-C

ABSTRACT

The present invention provides polynucleotides that encode the chemokine receptors 88-2B or 88C and materials and methods for the recombinant production of these two chemokine receptors. Also provided are assays utilizing the polynucleotides which facilitate the identification of ligands and modulators of the chemokine receptors. Receptor fragments, ligands, modulators, and antibodies are useful in the detection and treatment of disease states associated with the chemokine receptors such as atherosclerosis, rheumatoid arthritis, tumor growth suppression, asthma, viral infection, AIDS, and other inflammatory conditions.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/575,967 filed Dec. 20, 1995.

FIELD OF THE INVENTION

The present invention relates generally to signal transduction pathways.More particularly, the present invention relates to chemokine receptors,nucleic acids encoding chemokine receptors, chemokine receptor ligands,modulators of chemokine receptor activity, antibodies recognizingchemokines and chemokine receptors, methods for identifying chemokinereceptor ligands and modulators, methods for producing chemokinereceptors, and methods for producing antibodies recognizing chemokinereceptors.

BACKGROUND OF THE INVENTION

Recent advances in molecular biology have led to an appreciation of thecentral role of signal transduction pathways in biological processes.These pathways comprise a central means by which individual cells in amulticellular organism communicate, thereby coordinating biologicalprocesses. See Springer, Cell 76:301-314 (1994), Table I for a model.One branch of signal transduction pathways, defined by the intracellularparticipation of guanine nucleotide binding proteins (G-proteins),affects a broad range of biological processes.

Lewin, GENES V 319-348 (1994) generally discusses G-protein signaltransduction pathways which involve, at a minimum, the followingcomponents: an extracellular signal (e.g., neurotransmitters, peptidehormones, organic molecules, light, or odorants), a signal-recognizingreceptor (G-protein-coupled receptor, reviewed in Probst et al., DNA andCell Biology 11:1-20 [1992] and also known as GPR or GPCR), and anintracellular, heterotrimeric GTP-binding protein, or G protein. Inparticular, these pathways have attracted interest because of their rolein regulating white blood cell or leukocyte trafficking.

Leukocytes comprise a group of mobile blood cell types includinggranulocytes (i.e., neutrophils, basophils, and eosinophils),lymphocytes, and monocytes. When mobilized and activated, these cellsare primarily involved in the body's defense against foreign matter.This task is complicated by the diversity of normal and pathologicalprocesses in which leukocytes participate. For example, leukocytesfunction in the normal inflammatory response to infection. Leukocytesare also involved in a variety of pathological inflammations. For asummary, see Schall et al., Curr. Opin. Immunol. 6:865-873 (1994).Moreover, each of these processes can involve unique contributions, indegree, kind, and duration, from each of the leukocyte cell types.

In studying these immune reactions, researchers initially concentratedon the signals acting upon leukocytes, reasoning that a signal would berequired to elicit any form of response. Murphy, Ann. Rev. Immunol.12:593-633 (1994) has reviewed members of an important group ofleukocyte signals, the peptide signals. One type of peptide signalcomprises the chemokines (chemoat-tractant cytokines), termedintercrines in Oppenheim et al., Ann. Rev. Immunol. 9:617-648 (1991). Inaddition to Oppenheim et al., Baggiolini et al., Advances in Immunol.55:97-179 (1994), documents the growing number of chemokines that havebeen identified and subjected to genetic and biochemical analyses.

Comparisons of the amino acid sequences of the known chemokines have ledto a classification scheme which divides chemokines into two groups: theα group characterized by a single amino acid separating the first twocysteines (CXC; N-terminus as referent), and the β group, where thesecysteines are adjacent (CC). See Baggiolini et al., supra. Correlationshave been found between the chemokines and the particular leukocyte celltypes responding to those signals. Schall et al., supra, has reportedthat the CXC chemokines generally affect neutrophils; the CC chemokinestend to affect monocytes, lymphocytes, basophils and eosinophils. Forexample, Baggiolini et al., supra, recited that RANTES, a CC chemokine,functions as a chemoattractant for monocytes, lymphocytes (i.e., memoryT cells), basophils, and eosinophils, but not for neutrophils, whileinducing the release of histamine from basophils.

Chemokines were recently shown by Cocchi, et. al., Science, 270:1811-1815 (1995) to be suppressors of HIV proliferation. Cocchi, et al.demonstrated that RANTES, MIP-1α, and MIP-1β suppressed HIV-1, HIV-2 andSIV infection of a CD4⁺ cell line designated PM1 and of primary humanperipheral blood mononuclear cells.

Recently, however, attention has turned to the cellular receptors thatbind the chemokines, because the extracellular chemokines seem tocontact cells indiscriminately, and therefore lack the specificityneeded to regulate the individual leukocyte cell types.

Murphy, supra, reported that the GPCR superfamily of receptors includesthe chemokine receptor family. The typical chemokine receptor structureincludes an extracellular chemokine-binding domain located near theN-terminus, followed by seven spaced regions of predominantlyhydrophobic amino acids capable of forming membrane-spanning α-helices.Between each of the α-helical domains are hydrophilic domains localized,alternately, in the intra- or extra-cellular spaces. These featuresimpart a serpentine conformation to the membrane-embedded chemokinereceptor. The third intracellular loop typically interacts withG-proteins. In addition, Murphy, supra, noted that the intracellularcarboxyl terminus is also capable of interacting with G-proteins.

The first chemokine receptors to be analyzed by molecular cloningtechniques were the two neutrophil receptors for human IL8, a CXCchemokine. Holmes et al., Science 253:1278-1280 (1991) and Murphy etal., Science 253:1280-1283 (1991), reported the cloning of these tworeceptors for IL8. Lee et al., J. Biol. Chem. 267:16283-16287 (1992),analyzed the cDNAs encoding these receptors and found 77% amino acididentity between the encoded receptors, with each receptor exhibitingfeatures of the G protein coupled receptor family. One of thesereceptors is specific for IL-8, while the other binds and signals inresponse to IL-8, gro/MGSA, and NAP-2. Genetic manipulation of the genesencoding IL-8 receptors has contributed to our understanding of thebiological roles occupied by these receptors. For example, Cacalano etal., Science 265:682-684 (1994) reported that deletion of the IL-8receptor homolog in the mouse resulted in a pleiotropic phenotypeinvolving lymphadenopathy and splenomegaly. In addition, a study ofmissense mutations described in Leong et al., J. Biol. Chem.269:19343-19348 (1994) revealed amino acids in the IL-8 receptor thatwere critical for IL-8 binding. Domain swapping experiments discussed inMurphy, supra, implicated the amino terminal extracellular domain as adeterminant of binding specificity.

Several receptors for CC chemokines have also been identified andcloned. CCCKR1 binds both MIP-1α and RANTES and causes intracellularcalcium ion flux in response to both ligands. Charo et al., Proc Natl.Acad. Sci. (USA) 91:2752-2756 (1994) reported that another CC chemokinereceptor, MCP-R1 (CCCKR2), is encoded by a single gene that produces twosplice variants which differ in their carboxyl terminal domains. Thisreceptor binds and responds to MCP-3 in addition to MCP-1.

A promiscuous receptor that binds both CXC and CC chemokines has alsobeen identified. This receptor was originally identified on red bloodcells and Horuk et al., Science 261:1182-1184 (1993) reports that itbinds IL-8, NAP-2, GROα, RANTES, and MCP-1. The erythrocyte chemokinereceptor shares about 25% identity with other chemokine receptors andmay help to regulate circulating levels of chemokines or aid in thepresentation of chemokines to their targets. In addition to bindingchemokines, the erythrocyte chemokine receptor has also been shown to bethe receptor for plasmodium vivax, a major cause of malaria (id.)Another G-protein coupled receptor which is closely related to chemokinereceptors, the platelet activating factor receptor, has also been shownto be the receptor for a human pathogen, the bacterium Streptococcuspneumoniae (Cundell et al., Nature 377:435-438 (1995)).

In addition to the mammalian chemokine receptors, two viral chemokinereceptor homologs have been identified. Ahuja et al., J. Biol. Chem.268:20691-20694 (1993) describes a gene product from Herpesvirus saimirithat shares about 30% identity with the IL-8 receptors and binds CXCchemokines. Neote et al., Cell, 72:415-425 (1993) reports that humancytomegalovirus contains a gene encoding a receptor sharing about 30%identity with the CC chemokine receptors which binds MIP-1α, MIP-1β,MCP-1, and RANTES. These viral receptors may affect the normal role ofchemokines and provide a selective pathological advantage for the virus.

Because of the broad diversity of chemokines and their activities, thereare numerous receptors for the chemokines. The receptors which have beencharacterized represent only a fraction of the total complement ofchemokine receptors. There thus remains a need in the art for theidentification of additional chemokine receptors. The availability ofthese novel receptors will provide tools for the development oftherapeutic modulators of chemokine or chemokine receptor function. Itis contemplated by the present invention that such modulators are usefulas therapeutics for the treatment of atherosclerosis, rheumatoidarthritis, tumor growth suppression, asthma, viral infections, and otherinflammatory conditions. Alternatively, fragments or variants of thechemokine receptors, or antibodies recognizing those receptors, arecontemplated as therapeutics.

SUMMARY OF THE INVENTION

The present invention provides purified and isolated nucleic acidsencoding chemokine receptors involved in leukocyte trafficking.Polynucleotides of the invention (both sense and anti-sense strandsthereof) include genomic DNAs, cDNAs, and RNAs, as well as completely orpartially synthetic nucleic acids. Preferred polynucleotides of theinvention include the DNA encoding the chemokine receptor 88-2B that isset out in SEQ ID NO:3, the DNA encoding the chemokine receptor 88C thatis set out in SEQ ID NO:1, and DNAs which hybridize to those DNAs understandard stringent hybridization conditions, or which would hybridizebut for the redundancy of the genetic code. Exemplary stringenthybridization conditions are as follows: hybridization at 42° C. in 50%formamide, 5×SSC, 20 mM sodium phosphate, pH 6.8 and washing in 0.2×SSCat 55° C. It is understood by those of skill in the art that variationin these conditions occurs based on the length and GC nucleotide contentof the sequences to be hybridized. Formulas standard in the art areappropriate for determining exact hybridization conditions. See Sambrooket al., §§9.47-9.51 in Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Alsocontemplated by the invention are polynucleotides encoding domains of88-2B or 88C, for example, polynucleotides encoding one or moreextracellular domains of either protein or other biologically activefragments thereof. 88-2B extracellular domains correspond to SEQ ID NO:3and SEQ ID NO:4 at amino acid residues 1-36, 93-107, 171-196, and263-284. The extracellular domains of 88-2B are encoded bypolynucleotide sequences corresponding to SEQ ID NO:3 at nucleotides362-469, 638-682, 872-949, and 1148-1213. Extracellular domains of 88Ccorrespond to SEQ ID NO:1 and SEQ ID NO:2 at amino acid residues 1-32,89-112, 166-191, and 259-280. The 88C extracellular domains are encodedby polynucleotide sequences that correspond to SEQ ID NO:1 atnucleotides 55-150, 319-390, 550-627, and 829-894. The invention alsocomprehends polynucleotides encoding intracellular domains of thesechemokine receptors. The intracellular domains of 88-2B include aminoacids 60-71, 131-151, 219-240, and 306-355 of SEQ ID NO:3 and SEQ IDNO:4. Those domains are encoded by polynucleotide sequencescorresponding to SEQ ID NO:3 at nucleotides 539-574, 752-814, 1016-1081,and 1277-1426, respectively. The 88C intracellular domains include aminoacid residues 56-67, 125-145, 213-235, and 301-352 of SEQ ID NO:1 andSEQ ID NO:2. The intracellular domains of 88C are encoded bypolynucleotide sequences corresponding to SEQ ID NO:1 at nucleotides220-255, 427-489, 691-759, and 955-1110. Peptides corresponding to oneor more of the extracellular or intracellular domains, or antibodiesraised against those peptides, are contemplated as modulators ofreceptor activities, especially ligand and G protein binding activitiesof the receptors.

The nucleotide sequences of the invention may also be used to designoligonucleotides for use as labeled probes to isolate genomic DNAsencoding 88-2B or 88C under stringent hybridization conditions (i.e., bySouthern analyses and Polymerase Chain Reaction methodologies).Moreover, these oligonucleotide probes can be used to detect particularalleles of the genes encoding 88-2B or 88C, facilitating both diagnosisand gene therapy treatments of disease states associated with particularalleles. In addition, these oligonucleotides can be used to alterchemokine receptor genetics to facilitate identification of chemokinereceptor modulators. Also, the nucleotide sequences can be used todesign antisense genetic elements of use in exploring or altering thegenetics and expression of 88-2B or 88C. The invention also comprehendsbiological replicas (i.e., copies of isolated DNAs made in vivo or invitro) and RNA transcripts of DNAs of the invention. Autonomouslyreplicating recombinant constructions such as plasmid, viral, andchromosomal (e.g., YAC) nucleic acid vectors effectively incorporating88-2B or 88C polynucleotides, and, particularly, vectors wherein DNAeffectively encoding 88-2B or 88C is operatively linked to one or moreendogenous or heterologous expression control sequences are alsoprovided.

The 88-2B and 88C receptors may be produced naturally, recombinantly orsynthetically. Host cells (prokaryotic or eukaryotic) transformed ortransfected with polynucleotides of the invention by standard methodsmay be used to express the 88-2B and 88C chemokine receptors. Beyond theintact 88-2B or 88C gene products, biologically active fragments of88-2B or 88C, analogs of 88-2B or 88C, and synthetic peptides derivedfrom the amino acid sequences of 88-2B, set out in SEQ ID NO:4, or 88C,set out in SEQ ID NO:2, are contemplated by the invention. Moreover, the88-2B or 88C gene product, or a biologically active fragment of eithergene product, when produced in a eukaryotic cell, may bepost-translationally modified (e.g., via disulfide bond formation,glycosylation, phosphorylation, myristoylation, palmitoylation,acetylation, etc.) The invention further contemplates the 88-2B and 88Cgene products, or biologically active fragments thereof, in monomeric,homomultimeric, or heteromultimeric conformations.

In particular, one aspect of the invention involves antibody productscapable of specifically binding to the 88-2B or 88C chemokine receptors.The antibody products are generated by methods standard in the art usingrecombinant 88-2B or 88C receptors, synthetic peptides or peptidefragments of 88-2B or 88C receptors, host cells expressing 88-2B or 88Con their surfaces, or 88-2B or 88C receptors purified from naturalsources as immunogens. The antibody products may include monoclonalantibodies or polyclonal antibodies of any source or subtype. Moreover,monomeric, homomultimeric, and heteromultimeric antibodies, andfragments thereof, are contemplated by the invention. Further, theinvention comprehends CDR-grafted antibodies, “humanized” antibodies,and other modified antibody products retaining the ability tospecifically bind a chemokine receptor.

The invention also contemplates the use of antibody products fordetection of the 88-2B or 88C gene products, their analogs, orbiologically active fragments thereof. For example, antibody productsmay be used in diagnostic procedures designed to reveal correlationsbetween the expression of 88-2B, or 88C, and various normal orpathological states. In addition, antibody products can be used todiagnose tissue-specific variations in expression of 88-2B or 88C, theiranalogs, or biologically active fragments thereof. Antibody productsspecific for the 88-2B and 88C chemokine receptors may also act asmodulators of receptor activities. In another aspect, antibodies to88-2B or 88C receptors are useful for therapeutic purposes.

Assays for ligands capable of interacting with the chemokine receptorsof the invention are also provided. These assays may involve directdetection of chemokine receptor activity, for example, by monitoring thebinding of a labeled ligand to the receptor. In addition, these assaysmay be used to indirectly assess ligand interaction with the chemokinereceptor. As used herein the term “ligand” comprises molecules which areagonists and antagonists of 88-2B or 88C, and other molecules which bindto the receptors.

Direct detection of ligand binding to a chemokine receptor may beachieved using the following assay. Test compounds (i.e., putativeligands) are detectably labeled (e.g., radioiodinated). The detectablylabeled test compounds are then contacted with membrane preparationscontaining a chemokine receptor of the invention. Preferably, themembranes are prepared from host cells expressing chemokine receptors ofthe invention from recombinant vectors. Following an incubation periodto facilitate contact between the membrane-embedded chemokine receptorsand the detectably labeled test compounds, the membrane material iscollected on filters using vacuum filtration. The detectable labelassociated with the filters is then quantitated. For example,radiolabels are quantitated using liquid scintillationspectrophotometry. Using this technique, ligands binding to chemokinereceptors are identified. To confirm the identification of a ligand, adetectably labeled test compound is exposed to a membrane preparationdisplaying a chemokine receptor in the presence of increasing quantitiesof the test compound in an unlabeled state. A progressive reduction inthe level of filter-associated label as one adds increasing quantitiesof unlabeled test compound confirms the identification of that ligand.

Agonists are ligands which bind to the receptor and elicit intracellularsignal transduction and antagonists are ligands which bind to thereceptor but do not elicit intracellular signal transduction. Thedetermination of whether a particular ligand is an agonist or anantagonist can be determined, for example, by assaying G protein-coupledsignal transduction pathways. Activation of these pathways can bedetermined by measuring intracellular ca⁺⁺ flux, phospholipase Cactivity or adenylyl cyclase activity, in addition to other assays (seeexamples 5 and 6).

As discussed in detail in the Examples herein, chemokines that bind tothe 88C receptor include RANTES, MIP-1α, and MIP-1β, and chemokines thatbind to the 88-2B receptor include RANTES.

In another aspect, modulators of the interaction between the 88C and88-2B receptors and their ligands are specifically contemplated by theinvention. Modulators of chemokine receptor function may be identifiedusing assays similar to those used for identifying ligands. The membranepreparation displaying a chemokine receptor is exposed to a constant andknown quantity of a detectably labeled functional ligand. In addition,the membrane-bound chemokine receptor is also exposed to an increasingquantity of a test compound suspected of modulating the activity of thatchemokine receptor. If the levels of filter-associated label correlatewith the quantity of test compound, that compound is a modulator of theactivity of the chemokine receptor. If the level of filter-associatedlabel increases with increasing quantities of the test compound, anactivator has been identified. In contrast, if the level offilter-associated label varies inversely with the quantity of testcompound, an inhibitor of chemokine receptor activity has beenidentified. Testing for modulators of receptor binding in this wayallows for the rapid screening of many putative modulators, as poolscontaining many potential modulators can be tested simultaneously in thesame reaction.

The indirect assays for receptor binding involve measurements of theconcentration or level of activity of any of the components found in therelevant signal transduction pathway. Chemokine receptor activationoften is associated with an intracellular Ca⁺⁺ flux. Cells expressingchemokine receptors may be loaded with a calcium-sensitive dye. Uponactivation of the expressed receptor, a Ca⁺⁺ flux would be renderedspectrophotometrically detectable by the dye. Alternatively, the Ca⁺⁺flux could be detected microscopically. Parallel assays, using eithertechnique, may be performed in the presence and absence of putativeligands. For example, using the microscopic assay for Ca⁺⁺ flux, RANTES,a CC chemokine, was identified as a ligand of the 88-2B chemokinereceptor. Those skilled in the art will recognize that these assays arealso useful for identifying and monitoring the purification ofmodulators of receptor activity. Receptor activators and inhibitors willactivate or inhibit, respectively, the interaction of the receptors withtheir ligands in these assays.

Alternatively, the association of chemokine receptors with G proteinsaffords the opportunity of assessing receptor activity by monitoring Gprotein activities. A characteristic activity of G proteins, GTPhydrolysis, may be monitored using, for example, ³²P-labeled GTP.

G proteins also affect a variety of other molecules through theirparticipation in signal transduction pathways. For example, G proteineffector molecules include adenylyl cyclase, phospholipase C, ionchannels, and phosphodiesterases. Assays focused on any of theseeffectors may be used to monitor chemokine receptor activity induced byligand binding in a host cell that is both expressing the chemokinereceptor of interest and contacted with an appropriate ligand. Forexample, one method by which the activity of chemokine receptors may bedetected involves measuring phospholipase C activity. In this assay, theproduction of radiolabeled inositol phosphates by host cells expressinga chemokine receptor in the presence of an agonist is detected. Thedetection of phospholipase activity may require cotransfection with DNAencoding an exogenous G protein. When cotransfection is required, thisassay can be performed by cotransfection of chimeric G protein DNA, forexample, Gqi5 (Conklin, et al., Nature 363:274-276 (1993), with 88-2B or88C DNA and detecting phosphoinositol production when the cotransfectedcell is exposed to an agonist of the 88-2B or 88C receptor. Thoseskilled in the art will recognize that assays focused on G-proteineffector molecules are also useful for identifying and monitoring thepurification of modulators of receptor activity. Receptor activators andinhibitors will activate or inhibit, respectively, the interaction ofthe receptors with their ligands in these assays.

Chemokines have been linked to many inflammatory diseases, such aspsoriasis, arthritis, pulmonary fibrosis and atherosclerosis. SeeBaggiolini et al., supra. Inhibitors of chemokine action may be usefulin treating these conditions. In one example, Broaddus et al., J. ofImmunol. 152:2960-2967 (1994), describes an antibody to IL-8 which caninhibit neutrophil recruitment in endotoxin-induced pleurisy, a model ofacute inflammation in rabbit lung. It is also contemplated that ligandor modulator binding to, or the activation of, the 88C receptor may beuseful in treatment of HIV infection and HIV related disease states.Modulators of chemokine binding to specific receptors contemplated bythe invention may include antibodies directed toward a chemokine or areceptor, biological or chemical small molecules, or synthetic peptidescorresponding to fragments of the chemokine or receptor.

Administration of compositions containing 88-2B or 88C modulators tomammalian subjects, for the purpose of monitoring or remediating normalor pathological immune reactions And viral infections includinginfection by retroviruses such as HIV-1, HIV-2 and SIV is contemplatedby the invention. In particular, the invention comprehends themitigation of inflammatory responses, abnormal hematopoietic processes,and viral infections by delivery of a pharmaceutically acceptablequantity of 88-2B or 88C chemokine receptor modulators. The inventionfurther comprehends delivery of these active substances inpharmaceutically acceptable compositions comprising carriers, diluents,or medicaments. The invention also contemplates a variety ofadministration routes. For example, the active substances may beadministered by the following routes: intravenous, subcutaneous,intraperitoneal, intramuscular, oral, anal (i.e., via suppositoryformulations), or pulmonary (i.e., via inhalers, atomizers, nebulizers,etc.)

In another aspect, the DNA sequence information provided by the presentinvention makes possible the development, by homologous recombination or“knockout” strategies [see, e.g. Kapecchi, Science, 244:1288-1292(1989)], of rodents that fail to express a functional 88C or 88-2Bchemokine receptor or that express a variant of the receptor.Alternatively, transgenic mice which express a cloned 88-2B or 88Creceptor can be prepared by well known laboratory techniques(Manipulating the Mouse Embryo: A Laboratory Manual, Brigid Hohan, FrankCostantini and Elizabeth Lacy, eds. (1986) Cold Spring Harbor LaboratoryISBN 0-87969-175-I). Such rodents are useful as models for studying theactivities of 88C or 88-2B receptors in vivo.

Other aspects and advantages of the present invention will becomeapparent to one skilled in the art upon consideration of the followingexamples.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate the invention. Example 1 describes theisolation of genomic DNAs encoding the 88-2B and 88C chemokinereceptors. Example 2 presents the isolation and sequencing of cDNAsencoding 88-2B and 88C. Example 3 provides a description of Northernanalyses revealing the expression patterns of the 88-2B and 88Creceptors in a variety of tissues. Example 4 details the recombinantexpression of the 88-2B and 88C receptors. Example 5 describes Ca⁺⁺ fluxassays, phosphoinositol hydrolysis assays, and binding assays for 88-2Band 88C receptor activity in response to a variety of potential ligands.Example 6 describes additional assays designed to identify 88-2B or 88Cligands or modulators.

EXAMPLE 1

Partial genomic clones encoding the novel chemokine receptor genes ofthis invention were isolated by PCR based on conserved sequences foundin previously identified genes and based on a clustering of thesechemokine receptor genes within the human genome. The genomic DNA wasamplified by standard PCR methods using degenerate oligonucleotideprimers.

Templates for PCR amplifications were members of a commerciallyavailable source of recombinant human genomic DNA cloned into YeastArtificial Chromosomes (i.e., YACs). (Research Genetics, Inc.,Huntsville, Ala., YAC Library Pools, catalog no. 95011 B). A YAC vectorcan accommodate inserts of 500-1000 kilobase pairs. Initially, pools ofYAC clone DNAs were screened by PCR using primers specific for the geneencoding CCCKR1. In particular, CCCKR(2)-5′, the sense strand primer(corresponding to the sense strand of CCCKR1), is presented in SEQ IDNO:15. Primer CCCKR(2)-5′ consisted of the sequence5′-CGTAAGCTTAGAGAAGCCGGGATGGGAA-3′, wherein the underlined nucleotidesare the translation start codon for CCCKR1. The anti-sense strand primerwas CCCKR-3′ (corresponding to the anti-sense strand of CCCKR1) and itssequence is presented in SEQ ID NO:16. The sequence of CCCKR-3′,5′-GCCTCTAGAGTCAGAGACCAGCAGA-3′, contains the reverse complement of theCCCKR1 translation stop codon (underlined). Pools of YAC clone DNAsyielding detectable PCR products (i.e., DNA bands upon gelelectrophoresis) identified appropriate sub-pools of YAC clones, basedon a proprietary identification scheme. (Research Genetics, Inc.,Huntsville, Ala.). PCR reactions were initiated with an incubation at94° C. for four minutes. Sequence amplifications were achieved using 33cycles of denaturation at 94° C. for one minute, annealing at 55° C. forone minute, and extension at 72° C. for two minutes.

The sub-pools of YAC clone DNAs were then subjected to a second round ofPCR reactions using the conditions, and primers, that were used in thefirst round of PCR. Results from sub-pool screenings identifiedindividual clones capable of supporting PCR reactions with theCCCKR-specific primers. One clone, 881F10, contained 640 kb of humangenomic DNA from chromosome 3p21 including the genes for CCCKR1 andCCCKR2, as determined by PCR and hybridization. An overlapping YACclone, 941A7, contained 700 kb of human genomic DNA and also containedthe genes for CCCKR1 and CCCKR2. Consequently, further mapping studieswere undertaken using these two YAC clones. Southern analyses revealedthat CCCKR1 and CCCKR2 were located within approximately 100 kb of oneanother.

The close proximity of the CCCKR1 and CCCKR2 genes suggested that novelrelated genes might be linked to CCCKR1 and CCCKR2. Using DNA from yeastcontaining YAC clones 881F10 and 941A7 as templates, PCR reactions wereperformed to amplify any linked receptor genes. Degenerateoligodeoxyribonucleotides were designed as PCR primers. Theseoligonucleotides corresponded to regions encoding the secondintracellular loop and the sixth transmembrane domain of CC chemokinereceptors, as deduced from aligned sequence comparisons of CCCKR1,CCCKR2, and V28. V28 was used because it is an orphan receptor thatexhibits the characteristics of a chemokine receptor; V28 has also beenmapped to human chromosome 3. Raport et al., Gene 163:295-299 (1995). Offurther note, the two splice variants of CCCKR2, CCCKR2A and CCCKR2B,are identical in the second intracellular loop and sixth transmembranedomain regions used in the analysis. The 5′ primer, designated V28degf2,contains an internal BamHI site (see below); its sequence is presentedin SEQ ID NO:5. The sequence of primer V28degf2 corresponds to DNAencoding the second intracellular loop region of the canonical receptorstructure. See Probst et al., supra. The 3′ primer, designated V28degr2,contains an internal HindIII site (see below); its sequence is presentedin SEQ ID NO:6. The sequence of primer V28degr2 corresponds to DNAencoding the sixth transmembrane domain of the canonical receptorstructure.

Amplified PCR DNA was subsequently digested with BamHI and HindIII togenerate fragments of approximately 390 bp, consistent with the fragmentsize predicted from inspection of the canonical sequence. Followingendonuclease digestion, these PCR fragments were cloned into pBluescript(Stratagene Inc., LaJolla, Calif.). A total of 54 cloned fragments weresubjected to automated nucleotide sequence analyses. In addition tosequences from CCCKR1 and CCCKR2, sequences from the two novel chemokinereceptor genes of the invention were identified. These two novelchemokine receptor genes were designated 88-2B and 88C.

Restriction endonuclease mapping and hybridization were utilized to mapthe relative positions of genes encoding the receptors 88C, 88-2B,CCCKR1, and CCCKR2. These four genes are closely linked, as the gene for88C is approximately 18 KBP from the CCCKR2 gene on human chromosome3p21.

EXAMPLE 2

Full-length 88-2B and 88C cDNAs were isolated from a macrophage cDNAlibrary by the following procedure. Initially, a cDNA library, describedin Tjoelker et al., Nature 374:549-553 (1995), was constructed inpRc/CMV (Invitrogen Corp., San Diego, Calif.) from human macrophagemRNA. The cDNA library was screened for the presence of 88-2B and 88CcDNA clones by PCR using unique primer pairs corresponding to 88-2B or88C. The PCR protocol involved an initial denaturation at 94° C. forfour minutes. Polynucleotides were then amplified using 33 cycles of PCRunder the following conditions: Denaturation at 94° C. for one minute,annealing at 55° C. for one minute, and extension at 72° C. for twominutes. The first primer specific for 88-2B was primer 88-2B-f1,presented in SEQ ID NO:11. It corresponds to the sense strand of SEQ IDNO:3 at nucleotides 844-863. The second PCR primer specific for the geneencoding 88-2B was primer 88-2B-r1, presented in SEQ ID NO:12; the88-2B-r1 sequence corresponds to the anti-sense strand of SEQ ID NO:3 atnucleotides 1023-1042. Similarly, the sequence of the first primerspecific for the gene encoding 88C, primer 88C-f1, is presented in SEQID NO:13 and corresponds to the sense strand of SEQ ID NO:1 atnucleotides 453-471. The second primer specific for the gene encoding88C is primer 88C-r3, presented in SEQ ID NO:14; the sequence of 88C-r3corresponds to the anti-sense strand of SEQ ID NO:1 at nucleotides744-763.

The screening identified clone 777, a cDNA clone of 88-2B. Clone 777contained a DNA insert of 1915 bp including the full length codingsequence of 88-2B as determined by the following criteria: the clonecontained a long open reading frame beginning with an ATG codon,exhibited a Kozak sequence, and had an in-frame stop codon upstream. TheDNA and deduced amino acid sequences of the insert of clone 777 arepresented in SEQ ID NO:3 and SEQ ID NO:4, respectively. The 88-2Btranscript was relatively rare in the macrophage cDNA library. Duringthe library screen, only three 88-2B clones were identified from anestimated total of three million clones.

Screening for cDNA clones encoding the 88C chemokine receptor identifiedclones 101 and 134 which appeared to contain the entire 88C codingregion, including a putative initiation codon. However, these cloneslacked the additional 5′ sequence needed to confirm the identity of theinitiation codon. The 88C transcript was relatively abundant in themacrophage cDNA Library. During the library screen, it was estimatedthat 88C was present at one per 3000 transcripts (in a total ofapproximately three million clones in the library).

RACE PCR (Rapid Amplification of cDNA Ends) was performed to extendexisting 88C clone sequences, thereby facilitating the accuratecharacterization of the 5′ end of the 88C cDNA. Human spleen5′-RACE-ready cDNA was purchased from Clontech Laboratories, Inc., PaloAlto, Calif., and used according to the manufacturer's recommendations.The cDNA had been made “5′-RACE-ready” by ligating an anchor sequence tothe 5′ ends of the cDNA fragments. The anchor sequence is complementaryto an anchor primer supplied by Clontech Laboratories, Inc., Palo Alto,Calif. The anchor sequence-anchor primer duplex polynucleotide containsan EcoRI site. Human spleen cDNA was chosen as template DNA becauseNorthern blots had revealed that 88C was expressed in this tissue. ThePCR reactions were initiated by denaturing samples at 94° C. for fourminutes. Subsequently, sequences were amplified using 35 cyclesinvolving denaturation at 94° C. for one minute, annealing at 60° C. for45 seconds, and extension at 72° C. for two minutes. The first round ofPCR was performed on reaction mixtures containing 2 μl of the5′-RACE-ready spleen cDNA, 1 μl of the anchor primer, and 1 μl of primer88c-r4 (100 ng/μl) in a total reaction volume of 50 μl. The 88C-specificprimer, primer 88c-r4 (5′-GATAAGCCTCACAGCCCTGTG-3′), is presented in SEQID NO:7. The sequence of primer 88c-r4 corresponds to the anti-sensestrand of SEQ ID NO:1 at nucleotides 745-765. A second round of PCR wasperformed on reaction mixtures including 1 μl of the first PCR reactionwith 1 μl of anchor primer and 1 μl of primer 88C-r1b (100 ng/μl)containing the following sequence (5′-GCTAAGCTTGATGACTATCTTTAATGTC-3′)and presented in SEQ ID NO:8. The sequence of primer 88C-r1b contains aninternal HindIII cloning site (underlined). The sequence 3′ of theHindIII site corresponds to the anti-sense strand of SEQ ID NO:1 atnucleotides 636-654. The resulting PCR product was digested with EcoRIand HindIII and fractionated on a 1% agarose gel. The approximately 700bp fragment was isolated and cloned into pBluescript. Clones with thelargest inserts were sequenced. Alternatively, the intact PCR productwas ligated into vector pCR using a commercial TA cloning kit(Invitrogen Corp., San Diego, Calif.) for subsequent nucleotide sequencedeterminations.

The 88-2B and 88C cDNAs were sequenced using the PRISM™ Ready ReactionDyeDeoxy™ Terminator Cycle Sequencing Kit (Perkin Elmer Corp., FosterCity, Calif.) and an Applied Biosystems 373A DNA Sequencer. The insertof clone 777 provided the double-stranded template for sequencingreactions used to determine the 88-2B cDNA sequence. The sequence of theentire insert of clone 777 was determined and is presented as the 88-2BcDNA sequence and deduced amino acid sequence in SEQ ID NO:3. Thesequence is 1915 bp in length, including 361 bp of 5′ untranslated DNA(corresponding to SEQ ID NO:3 at nucleotides 1-361), a coding region of1065 bp (corresponding to SEQ ID NO:3 at nucleotides 362-1426), and 489bp of 3′ untranslated DNA (corresponding to SEQ ID NO:3 at nucleotides1427-1915). The 88-2B genomic DNA, described in Example 1 above,corresponds to SEQ ID NO:3 at nucleotides 746-1128. The 88C cDNAsequence, and deduced amino acid sequence, is presented in SEQ ID NO:1.The 88C cDNA sequence is a composite of sequences obtained from RACE-PCRcDNA, clone 134, and clone 101. The RACE-PCR cDNA was used as asequencing template to determine nucleotides 1-654 in SEQ ID NO:1,including the unique identification of 9 bp of 5′ untranslated cDNAsequence in SEQ ID NO:1 at nucleotides 1-9. The sequence obtained fromthe RACE PCR cDNA confirmed the position of the first methionine codonat nucleotides 55-57 in SEQ ID NO:1, and supported the conclusion thatclone 134 and clone 101 contained full-length copies of the 88C codingregion. Clone 134 contained 45 bp of 5′ untranslated cDNA (correspondingto SEQ ID NO:1 at nucleotides 10-54), the 1056 bp 88C coding region(corresponding to SEQ ID NO:1 at nucleotides 55-1110), and 492 bp of 3′untranslated cDNA (corresponding to SEQ ID NO:1 at nucleotides1111-1602). Clone 101 contained 25 bp of 5′ untranslated cDNA(corresponding to SEQ ID NO:1 at nucleotides 30-54), the 1056 bp 88Ccoding region (corresponding to SEQ ID NO:1 at nucleotides 55-1110), and2273 bp of 3′ untranslated cDNA (corresponding to SEQ ID NO:1 atnucleotides 1111-3383). The 88C genomic DNA described in Example 1above, corresponds to SEQ ID NO:1 at nucleotides 424-809.

The deduced amino acid sequences of 88-2B and 88C revealedhydrophobicity profiles characteristic of GPCRs, including sevenhydrophobic domains corresponding to GPCR transmembrane domains.Sequence comparisons with other GPCRs also revealed a degree ofidentity. Significantly, the deduced amino acid sequences of both 88-2Band 88C had highest identity with the sequences of the chemokinereceptors. Table 1 presents the results of these amino acid sequencecomparisons.

TABLE 1 Chemokine Receptors 88-2B 88C IL-8RA  30%  30% IL-8RB  31%  30%CCCKR1  62%  54% CCCKR2A  46%  66% CCCKR2B  50%  72% 88-2B 100%  50%88-C  50% 100%

Table 1 shows that 88-2B is most similar to CCCKR1 (62% identical at theamino acid level) and 88C is most similar to CCCKR2 (72% identical atthe amino acid level).

The deduced amino acid sequences of 88-2B and 88C also reveal theintracellular and extracellular domains characteristic of GPCRs. The88-2B extracellular domains correspond to the amino acid sequenceprovided in SEQ ID NO:3, and SEQ ID NO:4, at amino acid residues 1-36,93-107, 171-196, and 263-284. The extracellular domains of 88-2B areencoded by polynucleotide sequences corresponding to SEQ ID NO:3 atnucleotides 362-469, 638-682, 872-949, and 1148-1213. Extracellulardomains of 88C include amino acid residues 1-32, 89-112, 166-191, and259-280 in SEQ ID NO:1 and SEQ ID NO:2. The 88C extracellular domainsare encoded by polynucleotide sequences that correspond to SEQ ID NO:1at nucleotides 55-150, 319-390, 550-627, and 829-894. The intracellulardomains of 88-2B include amino acids 60-71, 131-151, 219-240, and306-355 of SEQ ID NO:3 and SEQ ID NO:4. Those domains are encoded bypolynucleotide sequences corresponding to SEQ ID NO:3 at nucleotides539-574, 752-814, 1016-1081, and 1277-1426, respectively. The 88Cintracellular domains include amino acid residues 56-67, 125-145,213-235, and 301-352 of SEQ ID NO:1 and SEQ ID NO:2. The intracellulardomains of 88C are encoded by polynucleotide sequences corresponding toSEQ ID NO:1 at nucleotides 220-255, 427-489, 691-759, and 955-1110.

EXAMPLE 3

The mRNA expression patterns of 88-2B and 88C were determined byNorthern blot analyses.

Northern blots containing immobilized poly A⁺ RNA from a variety ofhuman tissues were purchased from Clontech Laboratories, Inc., PaloAlto, Calif. In particular, the following tissues were examined: heart,brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, colon and peripheralblood leukocytes.

A probe specific for 88-2B nucleotide sequences was generated from cDNAclone 478. The cDNA insert in clone 478 contains sequence correspondingto SEQ ID NO:3 at nucleotides 641-1915. To generate a probe, clone 478was digested and the insert DNA fragment was isolated following gelelectrophoresis. The isolated insert fragment was then radiolabeled with³²p-labeled nucleotides, using techniques known in the art.

A probe specific for 88C nucleotide sequences was generated by isolatingand radiolabeling the insert DNA fragment found in clone 493. The insertfragment from clone 493 contains sequence corresponding to SEQ ID NO:1at nucleotides 421-1359. Again, conventional techniques involving³²p-labeled nucleotides were used to generate the probe.

Northern blots probed with 88-2B revealed an approximately 1.8 kb mRNAin peripheral blood leukocytes. The 88C Northerns showed anapproximately 4 kb mRNA in several human tissues, including a strongsignal when probing spleen or thymus tissue and less intense signalswhen analyzing mRNA from peripheral blood leukocytes and smallintestine. A relatively weak signal for 88C was detected in lung tissueand in ovarian tissue.

The expression of 88C in human T-cells and in hematopoietic cell lineswas also determined by Northern blot analysis. Levels of 88C in CD4⁺ andCD8⁺ T-cells were very high. The transcript was present at relativelyhigh levels in myeloid cell lines THP1 and HL-60 and also found in the Bcell line Jijoye. In addition, the cDNA was a relatively abundanttranscript in a human macrophage cDNA library based on PCR amplificationof library subfractions.

EXAMPLE 4

The 88-2B and 88C cDNAs were expressed by recombinant methods inmammalian cells.

For transient transfection experiments, 88C was subcloned into themammalian cell expression vector pBJ1 (Ishi, K. et. al., J. Biol. Chem270:16435-16440 (1995). The construct included sequences encoding aprolactin signal sequence for efficient cell surface expression and aFLAG epitope at the amino terminus of 88C to facilitate detection of theexpressed protein. The FLAG epitope consists of the sequence “DYKDDDD.”COS-7 cells were transiently transfected with the 88C expression plasmidusing Lipofectamine (Life Technology, Inc., Grand Island, N.Y.)following the manufacturer's instructions. Briefly, cells were seeded in24-well plates at a density of 4×10⁴ cells per well and grown overnight.The cells were then washed with PBS, and 0.3 mg of DNA mixed with 1.5 μlof lipofectamine in 0.25 ml of Opti-MEM was added to each well. After 5hours at 37° C., the medium was replaced with medium containing 10% FCS.quantitative ELISA confirmed that 88C was expressed at the cell surfacein transiently transfected COS-7 cells using the M1 antibody specificfor the FLAG epitope (Eastman Co., New Haven, Conn.).

The FLAG-tagged 88C receptor was also stably transfected into HEK-293cells, a human embryonic kidney cell line, using transfection reagentDOTAP(N-[1-[(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummethylsulfate,Boehringer-Mannheim,Inc.,Indianapolis,Ind.)according to themanufacturer's recommendations. Stable lines were selected in thepresence of the drug G418. The transfected HEK-293 cells were evaluatedfor expression of 88C at the cell surface by ELISA, using the M1antibody to the FLAG epitope. ELISA showed that 88C tagged with the FLAGepitope was expressed at the cell surface of stably transformed HEK-293Cells.

The 88-2B and 88C cDNAs were used to make stable HEK-293 transfectants.The 88-2B receptor cDNA was cloned behind the cytomegalovirus promoterin pRc/CMV (Invitrogen Corp., San Diego, Calif.) using a PCR-basedstrategy. The template for the PCR reaction was the cDNA insert in clone777. The PCR primers were 88-2B-3 (containing an internal XbaI site) and88-2B-5 (containing an internal HindIII site). The nucleotide sequenceof primer 88-2B-3 is presented in SEQ ID NO:9; the nucleotide sequenceof primer 88-2B-5 is presented in SEQ ID NO:10. An 1104 bp region ofcDNA was amplified. Following amplification, the DNA was digested withXbaI and HindIII and cloned into similarly digested pRc/CMV. Theresulting plasmid was named 777XP2, which contains 18 bp of 5′untranslated sequence, the entire coding region of 88-2B, and 3 bp of 3′untranslated sequence. For the 88C sequence, the full-length cDNA insertin clone 134 was not further modified before transfecting HEK-293 cells.

To create stably transformed cell lines, the pRc/CMV recombinant cloneswere transfected using transfection reagent DOTAP(N-[1-[(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammoniummethylsulfate,Boehringer-Mannheim, Inc., Indianapolis, Ind.) according to themanufacturer's recommendations, into HEK-293 cells, a human embryonickidney cell line. Stable lines were selected in the presence of the drugG418. Standard screening procedures (i.e., Northern blot analyses) wereperformed to identify stable cell lines expressing the highest levels of88-2B and 88C mRNA.

EXAMPLE 5

A. Ca⁺⁺ Flux Assays

To analyze polypeptide expression, a functional assay for chemokinereceptor activity was employed. A common feature of signalling throughthe known chemokine receptors is that signal transduction is associatedwith the release of intracellular calcium cations. Therefore,intracellular Ca⁺⁺ concentration in the transfected HEK-293 cells wasassayed to determine whether the 88-2B or 88C receptors responded to anyof the known chemokines.

HEK-293 cells, stably transfected with 88-2B, 88C (without the FLAGepitope sequence), or a control coding region (encoding IL8R or CCCKR2,see below) as described above, were grown in T75 flasks to approximately90% confluence in MEM+10% serum. Cells were then washed, harvested withversene (0.6 mM EDTA, 10 mM Na₂HPO₄, 0.14 M NaCl, 3 mM KCl, and 1 mMglucose), and incubated in MEM+10% serum+1 μM Fura-2 AM (MolecularProbes, Inc., Eugene, Oreg.) for 30 minutes at room temperature. Fura-2AM is a Ca⁺⁺-sensitive dye. The cells were resuspended in Dulbecco'sphosphate-buffered saline containing 0.9 mM CaCl₂ and 0.5 mM MgCl₂(D-PBS) to a concentration of approximately 10⁷ cells/ml and changes influorescence were monitored using a fluorescence spectrophotometer(Hitachi Model F-4010). Approximately 10⁶ cells were suspended in 1.8 mlD-PBS in a cuvette maintained at 37° C. Excitation wavelengthsalternated between 340 and 380 nm at 4 second intervals; the emissionwavelength was 510 nm. Test compositions were added to the cuvette viaan injection port; maximal Ca⁺⁺ flux was measured upon the addition ofionomycin.

Positive responses were observed in cells expressing IL-8RA whenstimulated with IL-8 and also when CCCKR2 was stimulated with MCP-1 orMCP-3. However, HEK-293 cells expressing either 88-2B or 88C failed toshow a flux in intracellular Ca⁺⁺ concentration when exposed to any ofthe following chemokines: MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β, IL8,NAP-2, gro/MGSA, IP-10, ENA-78, or PF-4. (Peprotech, Inc., Rocky Hill,N.J.).

Using a more sensitive assay, a Ca⁺⁺ flux response to RANTES wasobserved microscopically in Fura-2 AM-loaded cells expressing 88-2B. Theassay involved cells and reagents prepared as described above. RANTES(Regulated on Activation, Normal T Expressed and Secreted) is a CCchemokine that has been identified as a chemoattractant and activator ofeosinophils. See Neote et al., supra. This chemokine also mediates therelease of histamine by basophils and has been shown to function as achemoattractant for memory T cells in vitro. Modulation of 88-2Breceptor activities is therefore contemplated to be useful in modulatingleukocyte activation.

FLAG tagged 88C receptor was expressed in HEK-293 cells and tested forchemokine interactions in the CA⁺⁺ flux assay. Cell surface expressionof 88C was confirmed by ELISA and by FACScan analysis using the M1antibody. The chemokines RANTES, MIP-1α, and MIP-1β all induced a Ca⁺⁺flux in 88C-transfected cells when added at a concentration of 100 nM.

Ca⁺⁺ flux assays can also be designed to identify modulators ofchemokine receptor binding. The preceding fluorimetric or microscopicassays are carried out in the presence of test compounds. If Ca⁺⁺ fluxis increased in the presence of a test compound, that compound is anactivator of chemokine receptor binding. In contrast, a diminished Ca⁺⁺flux identifies the test compound as an inhibitor of chemokine receptorbinding.

B. Phosphoinositol Hydrolysis

Another assay for ligands or modulators involves monitoringphospholipase C activity, as described in Hung et al., J. Biol. Chem.116:827-832 (1992). Initially, host cells expressing a chemokinereceptor are loaded with ³H-inositol for 24 hours. Test compounds (i.e.,potential ligands) are then added to the cells and incubated at 37° C.for 15 minutes. The cells are then exposed to 20 mM formic acid tosolubilize and extract hydrolyzed metabolites of phosphoinositolmetabolism (i.e., the products of phospholipase C-mediated hydrolysis).The extract is subjected to anion exchange chromatography using an AG1X8anion exchange column (formate form). Inositol phosphates are elutedwith 2 M ammonium formate/0.1 M formic acid and the ³H associated withthe compounds is determined using liquid scintillationspectrophotometry. The phospholipase C assay can also be exploited toidentify modulators of chemokine receptor activity. The aforementionedassay is performed as described, but with the addition of a potentialmodulator. Elevated levels of detectable label would indicate themodulator is an activator; depressed levels of the label would indicatethe modulator is an inhibitor of chemokine receptor activity.

The phospholipase C assay was performed to identify chemokine ligands ofthe FLAG-tagged 88C receptor. Approximately 24 hours after transfection,COS-7 cells expressing 88C were labeled for 20-24 hours withmyo-[2-³H]inositol (1 μCi/ml) in inositol-free medium containing 10%dialyzed FCS. Labeled cells were washed with inositol-free DMEMcontaining 10 mM LiCl and incubated at 37° C. for 1 hour withinositol-free DMEM containing 10 mM LiCl and one of the followingchemokines: RANTES, MIP-1β, MIP-1α, MCP-1, IL-8, or the murine MCP-1homolog JE. Inositol phosphate (IP) formation was assayed as describedin the previous paragraph. After incubation with chemokines, the mediumwas aspirated and cells were lysed by addition of 0.75 ml of ice-cold 20mM formic acid (30 min). Supernatant fractions were loaded onto AG1-X8Dowex columns (Biorad, Hercules, Calif.), followed by immediate additionof 3 ml of 50 mM NH₄OH. The columns were then washed with 4 ml of 40 mMammonium formate, followed by elution with 2 M ammonium formate. Totalinositol phosphates were quantitated by counting beta-emissions.

Because it has been shown that some chemokine receptors, such as IL8RAAND IL8RB, require contransfection with an exogenous G protein beforesignalling can be detected in COS-7 cells, the 88C receptor wasco-expressed with the chimeric G protein Gqi5 (Conklin, et al., Nature363:274-276, (1993). Gqi5 ia a G protein which has the carboxyl terminalfive amino acids of Gi (which bind to the receptor) spliced onto Gαq.Co-transfection with Gqi5 significantly potentiates signaling by CCCKR1and CCKR2B. Co-transfection with Gqi5 revealed that 88C signaled well inresponse to RANTES, MIP-1β, and MIP-1α, but not in response to MCP-1,IL-8 or the murine MCP-1 homologue JE. Dose-response curves revealedEC₅₀ values of 1 nM for RANTES, 6 nM for MIP-1β, and 22 nM for MIP-1α.

88C is the first cloned human receptor with a signaling response toMIP-1β. Compared with other CC chemokines, MIP-1β clearly has a uniquecellular activation pattern. It appears to activate T cells but notmonocytes (Baggiolini et al., Supra) which is consistent with receptorstimulation studies. For example, while MIP-1β binds to CCCKR1, it doesnot induce calcium flux (Neote et al., Supra). In contrast, MIP-1α andRANTES bind to and causes signalling in CCCKR1 and CCCKR5 (RANTES alsocauses activation of CCCKR3). MIP-1β thus appears to be much moreselective than other chemokines of the CC chemokine family. Suchselectivity is of therapeutic significance because a specific beneficialactivity can be stimulated (such as suppression of HIV infection)without stimulating multiple leukocyte populations which results ingeneral pro-inflammatory activities.

C. Binding Assays

Another assay for receptor interaction with chemokines was amodification of the binding asssay described by Ernst et al. J. Immunol.152:3541-3549 (1994). MIP-1β as labeled using the Bolton and Hunterreagent (di-iodide, NEN, Wilmington, Del.), according to themanufacturer's instructions. Unconjugated iodide was separated fromlabeled protein by elution using a PD-10 column (Pharmacia) equilibratedwith PBS and BSA (1% w/v). The specific activity was typically 2200Ci/mmole. Equilibrium binding was performed by adding ¹²⁵I-labeledligand with or without a 100-fold excess of unlabeled ligand, to 5×10⁵HEK-293 cells transfected with 88C tagged with the FLAG epitope inpolypropylene tubes in a total volume of 300 μl (50 mM HEPES pH 7.4, 1mM CaCl₂, MgCl₂, 0.5% BSA) and incubating for 90 minutes at 27° C. withshaking at 150 rpm. The cells were collected, using a Skatron cellharvester (Skatron Instruments Inc., Sterling, Va.), on glass fiberfilters presoaked in 0.3% polyethyleneimine and 0.2% BSA. After washing,the filters were removed and bound ligand was quantitated by countinggamma emissions. Ligand binding by competition with unlabeled ligand wasdetermined by incubation of 5×10⁵ transfected cells (as above) with 1.5nM of radiolabeled ligand and the indicated concentrations of unlabeledligand. The samples were collected, washed and counted as above. Thedata was analyzed using the curve-fitting program Prism (GraphPad Inc.,San Diego, Calif.) and the iterative non-linear regression program,LIGAND (PM220).

In equilibrium binding assays, 88C receptor bound radiolabeled MIP-1β ina specific and saturable manner. Analysis of this binding data by themethod of Scatchard revealed a dissociation constant (Kd) of 1.6 nM.Competition binding assays using labeled MIP-1β revealed high-affinitybinding of MIP-1β (IC₅₀=7.4 nM), RANTES (IC₅₀=6.9 nM), and MIP-1α(IC₅₀=7.4 nM), consistent with the signaling data obtained intransiently transfected COS-7 cells as discussed in section B above.

In view of these results, the present invention contemplates thatactivation of or ligand binding to the 88C receptor may provide aprotective role in HIV infection because the chemokines MIP-1β, RANTES,and MIP-1α have been shown to inhibit replication of HIV-1 and HIV-2 inhuman peripheral blood mononuclear cells and PM1 cells (Cocchi, et. al.,supra).

Recently, it has been reported that the orphan G protein-coupledreceptor, fusin, can act as a co-receptor for HIV entry. Fusin incombination with CD4, the primary HIV receptor, apparently facilitatesHIV infection of cultured T cells (Feng, et al., Science 272:872-877(1996). Based upon the homology of fusin to chemokine receptors and thechemokine binding profile of 88C, and because 88C is constitutivelyexpressed in T cells and abundant in macrophages, 88C is likely to beinvolved in viral and HIV infection.

Whether 88C and 88-2B function as a co-receptor for HIV can bedetermined by co-transfecting cells with CD4 and 88C or 88-2B andchallenging the co-transfected cells with HIV. Only cells expressingboth CD4 and a functional co-receptor for HIV will become infected. HIVinfection can be determined by several methods. ELISAs which test forexpression of HIV antigens are commercially available, for exampleCoulter HIV-1 _(p)24 antigen assay (U.S. Pat. No. 4,886,742), CoulterCorp., 11800 SW 147th Ave., Miami, Fla. 33196. Alternatively, the testcells can be engineered to express a reporter gene such as LACZ attachedto the HIV LTR promoter (Kimpton et al., J. Virol. 66:2232-2239 (1992)).In this method, cells that are infected with HIV are detected by acolorimetric assay.

Alternatively, in another experimental method which does not require theuse of live virus, cell lines co-expressing 88C or 88-2B, CD4 and a LACZreporter gene are mixed with a cell line co-expressing the HIV envelopeglycoprotein (ENV) and a transcription factor for the reporter geneconstruct (Nussbaum, et al., 1994 J. Virol. 68:5411). Cells expressing afunctional co-receptor for HIV will fuse with the ENV expressing cellsand thereby allow expression of the reporter gene. In this method,detection of reporter gene product by colorimetric assay indicates that88C or 88-2B function as a co-receptor for HIV.

The mechanism by which chemokines inhibit viral infection has not yetbeen elucidated. One possible mechanism involves activation of thereceptor by binding of a chemokine. The binding of the chemokine leadsto signal transduction events in the cell that renders the cellresistant to viral infection and/or prevents replication of the virus inthe cell. Similar to interferon induction, the cell may differentiatesuch that it is resistant to viral infection, or an antiviral state isestablished. Alternatively, a second mechanism involves directinterference with viral entry into cells by blocking access of viralenvelope glycoproteins to the co-receptor by chemokine binding. In thismechanism, G-protein signalling is not required for chemokinesuppression of HIV infection.

To distinguish between two mechanisms by which 88C or 88-2B may functionas co-receptors for viral or HIV infection, chemokine binding to thereceptor is uncoupled from signal transduction and the effect of thechemokine on suppression of viral infection is determined.

Ligand binding can be uncoupled from signal transduction by the additionof compounds which inhibit G-protein mediated signaling. These compoundsinclude, for example, pertussis toxin and cholera toxin. In addition,downstream effector polypeptides can be inhibited by other compoundssuch as wortmannin. If G-protein signalling is involved in suppressionof viral infection, the addition of such compounds would preventsuppression of viral infection by the chemokine. Alternatively, keyresidues or receptor domains of 88C or 88-2B receptor required forG-protein coupling can be altered or deleted such that G-proteincoupling is altered or destroyed but chemokine binding is not affected.

Under these conditions, if chemokines are unable to suppress viral orHIV infection, then signaling through a G-protein is required forsuppression of viral or HIV infection. If however, chemokines are ableto suppress viral infection, then G-protein signaling is not requiredfor chemokine suppression of viral infection and the protective effectsof chemokines may be due to the chemokine blocking the availability ofthe receptor for the virus.

Another approach involves the use of antibodies directed against 88C or88-2B. Antibodies which bind to 88C or 88-2B which can be shown not toelicit G-protein signaling may block access to the chemokine or viralbinding site of the receptor. If in the presence of antibodies to 88C or88-2B, viral infection is suppressed, then the mechanism of theprotective effects of chemokines is blocking viral access to itsreceptor. Feng, et al. Reported that antibodies to the amino terminus ofthe fusin receptor suppressed HIV infection (Feng, et al., 1996).

EXAMPLE 6

Additional methods may be used to identify ligands and modulators of thechemokine receptors of the invention.

In one embodiment, the invention comprehends a direct assay for ligands.Detectably labeled test compounds are exposed to membrane preparationspresenting chemokine receptors in a functional conformation. Forexample, HEK-293 cells, or tissue culture cells, are transfected with anexpression vehicle encoding a chemokine receptor. A membrane preparationis then made from the transfected cells expressing the chemokinereceptor. The membrane preparation is exposed to ¹²⁵I-labeled testcompounds (e.g., chemokines) and incubated under suitable conditions(e.g., 10 minutes at 37° C.). The membranes, with any bound testcompounds, are then collected on a filter by vacuum filtration andwashed to remove unbound test compounds. The radioactivity associatedwith the bound test compound is then quantitated by subjecting thefilters to liquid scintillation spectrophotometry. The specificity oftest compound binding may be confirmed by repeating the assay in thepresence of increasing quantities of unlabeled test compound and notingthe level of competition for binding to the receptor. These bindingassays can also identify modulators of chemokine receptor binding. Thepreviously described binding assay may be performed with the followingmodifications. In addition to detectably labeled test compound, apotential modulator is exposed to the membrane preparation. An increasedlevel of membrane-associated label indicates the potential modulator isan activator; a decreased level of membrane-associated label indicatesthe potential modulator is an inhibitor of chemokine receptor binding.

In another embodiment, the invention comprehends indirect assays foridentifying receptor ligands that exploit the coupling of chemokinereceptors to G proteins. As reviewed in Linder et al., Sci. Am.,267:56-65 (1992), during signal transduction, an activated receptorinteracts with a G protein, in turn activating the G protein. The Gprotein is activated by exchanging GDP for GTP. Subsequent hydrolysis ofthe G protein-bound GTP deactivates the G protein. One assay for Gprotein activity therefore monitors the release of ³²P_(i) from[γ-³²P]-GTP. For example, approximately 5×10⁷ HEK-293 cells harboringplasmids of the invention are grown in MEM+10% FCS. The growth medium issupplemented with 5 mCi/ml [³²P]-sodium phosphate for 2 hours touniformly label nucleotide pools. The cells are subsequently washed in alow-phosphate isotonic buffer. One aliquot of washed cells is thenexposed to a test compound while a second aliquot of cells is treatedsimilarly, but without exposure to the test compound. Following anincubation period (e.g., 10 minutes), cells are pelleted, lysed andnucleotide compounds fractionated using thin layer chromatographydeveloped with 1 M LiCl. Labeled GTP and GDP are identified byco-developing known standards. The labeled GTP and GDP are thenquantitated by autoradiographic techniques that are standard in the art.Relatively high levels of ³²P-labeled GDP identify test compounds asligands. This type of GTP hydrolysis assay is also useful for theidentification of modulators of chemokine receptor binding. Theaforementioned assay is performed in the presence of a potentialmodulator. An intensified signal resulting from a relative increase inGTP hydrolysis, producing ³²P-labeled GDP, indicates a relative increasein receptor activity. The intensified signal therefore identifies thepotential modulator as an activator. Conversely, a diminished relativesignal for ³²P-labeled GDP, indicative of decreased receptor activity,identifies the potential modulator as an inhibitor of chemokine receptorbinding.

The activities of G protein effector molecules (e.g., adenylyl cyclase,phospholipase C, ion channels, and phosphodiesterases) are also amenableto assay. Assays for the activities of these effector molecules havebeen previously described. For example, adenylyl cyclase, whichcatalyzes the synthesis of cyclic adenosine monophosphate (cAMP), isactivated by G proteins. Therefore, ligand binding to a chemokinereceptor that activates a G protein, which in turn activates adenylylcyclase, can be detected by monitoring cAMP levels in a recombinant hostcell of the invention. Implementing appropriate controls understood inthe art, an elevated level of intracellular cAMP can be attributed to aligand-induced increase in receptor activity, thereby identifying aligand. Again using controls understood in the art, a relative reductionin the concentration of cAMP would indirectly identify an inhibitor ofreceptor activity. The concentration of cAMP can be measured by acommercial enzyme immunoassay. For example, the BioTrak Kit providesreagents for a competitive immunoassay. (Amersham, Inc., ArlingtonHeights, Ill.). Using this kit according to the manufacturer'srecommendations, a reaction is designed that involves competingunlabeled cAMP with cAMP conjugated to horseradish peroxidase. Theunlabeled cAMP may be obtained, for example, from activated cellsexpressing the chemokine receptors of the invention. The two compoundscompete for binding to an immobilized anti-cAMP antibody. After thecompetition reaction, the immobilized horseradish peroxidase-cAMPconjugate is quantitated by enzyme assay using atetramethylbenzidine/H₂O₂ single-pot substrate with detection of coloredreaction products occurring at 450 nm. The results provide a basis forcalculating the level of unlabeled cAMP, using techniques that arestandard in the art. In addition to identifying ligands binding tochemokine receptors, the cAMP assay can also be used to identifymodulators of chemokine receptor binding. Using recombinant host cellsof the invention, the assay is performed as previously described, withthe addition of a potential modulator of chemokine receptor activity. Byusing controls that are understood in the art, a relative increase ordecrease in intracellular cAMP levels reflects the activation orinhibition of adenylyl cyclase activity. The level of adenylyl cyclaseactivity, in turn, reflects the relative activity of the chemokinereceptor of interest. A relatively elevated level of chemokine receptoractivity identifies an activator; a relatively reduced level of receptoractivity identifies an inhibitor of chemokine receptor activity.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the appended claims should be placed on the invention.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 16(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 3383 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 55..1110 (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88C polynucleotide and aminoacid sequences” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGAAGAGCTGAGACATCCGT TCCCCTACAA GAAACTCTCC CCGGGTGGAA CAAG ATG 57 Met 1 GAT TATCAA GTG TCA AGT CCA ATC TAT GAC ATC AAT TAT TAT ACA TCG 105 Asp Tyr GlnVal Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr Ser 5 10 15 GAG CCC TGCCAA AAA ATC AAT GTG AAG CAA ATC GCA GCC CGC CTC CTG 153 Glu Pro Cys GlnLys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu Leu 20 25 30 CCT CCG CTC TACTCA CTG GTG TTC ATC TTT GGT TTT GTG GGC AAC ATG 201 Pro Pro Leu Tyr SerLeu Val Phe Ile Phe Gly Phe Val Gly Asn Met 35 40 45 CTG GTC ATC CTC ATCCTG ATA AAC TGC AAA AGG CTG AAG AGC ATG ACT 249 Leu Val Ile Leu Ile LeuIle Asn Cys Lys Arg Leu Lys Ser Met Thr 50 55 60 65 GAC ATC TAC CTG CTCAAC CTG GCC ATC TCT GAC CTG TTT TTC CTT CTT 297 Asp Ile Tyr Leu Leu AsnLeu Ala Ile Ser Asp Leu Phe Phe Leu Leu 70 75 80 ACT GTC CCC TTC TGG GCTCAC TAT GCT GCC GCC CAG TGG GAC TTT GGA 345 Thr Val Pro Phe Trp Ala HisTyr Ala Ala Ala Gln Trp Asp Phe Gly 85 90 95 AAT ACA ATG TGT CAA CTC TTGACA GGG CTC TAT TTT ATA GGC TTC TTC 393 Asn Thr Met Cys Gln Leu Leu ThrGly Leu Tyr Phe Ile Gly Phe Phe 100 105 110 TCT GGA ATC TTC TTC ATC ATCCTC CTG ACA ATC GAT AGG TAC CTG GCT 441 Ser Gly Ile Phe Phe Ile Ile LeuLeu Thr Ile Asp Arg Tyr Leu Ala 115 120 125 GTC GTC CAT GCT GTG TTT GCTTTA AAA GCC AGG ACG GTC ACC TTT GGG 489 Val Val His Ala Val Phe Ala LeuLys Ala Arg Thr Val Thr Phe Gly 130 135 140 145 GTG GTG ACA AGT GTG ATCACT TGG GTG GTG GCT GTG TTT GCG TCT CTC 537 Val Val Thr Ser Val Ile ThrTrp Val Val Ala Val Phe Ala Ser Leu 150 155 160 CCA GGA ATC ATC TTT ACCAGA TCT CAA AAA GAA GGT CTT CAT TAC ACC 585 Pro Gly Ile Ile Phe Thr ArgSer Gln Lys Glu Gly Leu His Tyr Thr 165 170 175 TGC AGC TCT CAT TTT CCATAC AGT CAG TAT CAA TTC TGG AAG AAT TTC 633 Cys Ser Ser His Phe Pro TyrSer Gln Tyr Gln Phe Trp Lys Asn Phe 180 185 190 CAG ACA TTA AAG ATA GTCATC TTG GGG CTG GTC CTG CCG CTG CTT GTC 681 Gln Thr Leu Lys Ile Val IleLeu Gly Leu Val Leu Pro Leu Leu Val 195 200 205 ATG GTC ATC TGC TAC TCGGGA ATC CTA AAA ACT CTG CTT CGG TGT CGA 729 Met Val Ile Cys Tyr Ser GlyIle Leu Lys Thr Leu Leu Arg Cys Arg 210 215 220 225 AAT GAG AAG AAG AGGCAC AGG GCT GTG AGG CTT ATC TTC ACC ATC ATG 777 Asn Glu Lys Lys Arg HisArg Ala Val Arg Leu Ile Phe Thr Ile Met 230 235 240 ATT GTT TAT TTT CTCTTC TGG GCT CCC TAC AAC ATT GTC CTT CTC CTG 825 Ile Val Tyr Phe Leu PheTrp Ala Pro Tyr Asn Ile Val Leu Leu Leu 245 250 255 AAC ACC TTC CAG GAATTC TTT GGC CTG AAT AAT TGC AGT AGC TCT AAC 873 Asn Thr Phe Gln Glu PhePhe Gly Leu Asn Asn Cys Ser Ser Ser Asn 260 265 270 AGG TTG GAC CAA GCTATG CAG GTG ACA GAG ACT CTT GGG ATG ACG CAC 921 Arg Leu Asp Gln Ala MetGln Val Thr Glu Thr Leu Gly Met Thr His 275 280 285 TGC TGC ATC AAC CCCATC ATC TAT GCC TTT GTC GGG GAG AAG TTC AGA 969 Cys Cys Ile Asn Pro IleIle Tyr Ala Phe Val Gly Glu Lys Phe Arg 290 295 300 305 AAC TAC CTC TTAGTC TTC TTC CAA AAG CAC ATT GCC AAA CGC TTC TGC 1017 Asn Tyr Leu Leu ValPhe Phe Gln Lys His Ile Ala Lys Arg Phe Cys 310 315 320 AAA TGC TGT TCTATT TTC CAG CAA GAG GCT CCC GAG CGA GCA AGC TCA 1065 Lys Cys Cys Ser IlePhe Gln Gln Glu Ala Pro Glu Arg Ala Ser Ser 325 330 335 GTT TAC ACC CGATCC ACT GGG GAG CAG GAA ATA TCT GTG GGC TTG 1110 Val Tyr Thr Arg Ser ThrGly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350 TGACACGGAC TCAAGTGGGCTGGTGACCCA GTCAGAGTTG TGCACATGGC TTAGTTTTCA 1170 TACACAGCCT GGGCTGGGGGTGGGGTGGGA GAGGTCTTTT TTAAAAGGAA GTTACTGTTA 1230 TAGAGGGTCT AAGATTCATCCATTTATTTG GCATCTGTTT AAAGTAGATT AGATCTTTTA 1290 AGCCCATCAA TTATAGAAAGCCAAATCAAA ATATGTTGAT GAAAAATAGC AACCTTTTTA 1350 TCTCCCCTTC ACATGCATCAAGTTATTGAC AAACTCTCCC TTCACTCCGA AAGTTCCTTA 1410 TGTATATTTA AAAGAAAGCCTCAGAGAATT GCTGATTCTT GAGTTTAGTG ATCTGAACAG 1470 AAATACCAAA ATTATTTCAGAAATGTACAA CTTTTTACCT AGTACAAGGC AACATATAGG 1530 TTGTAAATGT GTTTAAAACAGGTCTTTGTC TTGCTATGGG GAGAAAAGAC ATGAATATGA 1590 TTAGTAAAGA AATGACACTTTTCATGTGTG ATTTCCCCTC CAAGGTATGG TTAATAAGTT 1650 TCACTGACTT AGAACCAGGCGAGAGACTTG TGGCCTGGGA GAGCTGGGGA AGCTTCTTAA 1710 ATGAGAAGGA ATTTGAGTTGGATCATCTAT TGCTGGCAAA GACAGAAGCC TCACTGCAAG 1770 CACTGCATGG GCAAGCTTGGCTGTAGAAGG AGACAGAGCT GGTTGGGAAG ACATGGGGAG 1830 GAAGGACAAG GCTAGATCATGAAGAACCTT GACGGCATTG CTCCGTCTAA GTCATGAGCT 1890 GAGCAGGGAG ATCCTGGTTGGTGTTGCAGA AGGTTTACTC TGTGGCCAAA GGAGGGTCAG 1950 GAAGGATGAG CATTTAGGGCAAGGAGACCA CCAACAGCCC TCAGGTCAGG GTGAGGATGG 2010 CCTCTGCTAA GCTCAAGGCGTGAGGATGGG AAGGAGGGAG GTATTCGTAA GGATGGGAAG 2070 GAGGGAGGTA TTCGTGCAGCATATGAGGAT GCAGAGTCAG CAGAACTGGG GTGGATTTGG 2130 TTTGGAAGTG AGGGTCAGAGAGGAGTCAGA GAGAATCCCT AGTCTTCAAG CAGATTGGAG 2190 AAACCCTTGA AAAGACATCAAGCACAGAAG GAGGAGGAGG AGGTTTAGGT CAAGAAGAAG 2250 ATGGATTGGT GTAAAAGGATGGGTCTGGTT TGCAGAGCTT GAACACAGTC TCACCCAGAC 2310 TCCAGGCTGT CTTTCACTGAATGCTTCTGA CTTCATAGAT TTCCTTCCCA TCCCAGCTGA 2370 AATACTGAGG GGTCTCCAGGAGGAGACTAG ATTTATGAAT ACACGAGGTA TGAGGTCTAG 2430 GAACATACTT CAGCTCACACATGAGATCTA GGTGAGGATT GATTACCTAG TAGTCATTTC 2490 ATGGGTTGTT GGGAGGATTCTATGAGGCAA CCACAGGCAG CATTTAGCAC ATACTACACA 2550 TTCAATAAGC ATCAAACTCTTAGTTACTCA TTCAGGGATA GCACTGAGCA AAGCATTGAG 2610 CAAAGGGGTC CCATATAGGTGAGGGAAGCC TGAAAAACTA AGATGCTGCC TGCCCAGTGC 2670 ACACAAGTGT AGGTATCATTTTCTGCATTT AACCGTCAAT AGGCAAAGGG GGGAAGGGAC 2730 ATATTCATTT GGAAATAAGCTGCCTTGAGC CTTAAAACCC ACAAAAGTAC AATTTACCAG 2790 CCTCCGTATT TCAGACTGAATGGGGGTGGG GGGGGCGCCT TAGGTACTTA TTCCAGATGC 2850 CTTCTCCAGA CAAACCAGAAGCAACAGAAA AAATCGTCTC TCCCTCCCTT TGAAATGAAT 2910 ATACCCCTTA GTGTTTGGGTATATTCATTT CAAAGGGAGA GAGAGAGGTT TTTTTCTGTT 2970 CTTTCTCATA TGATTGTGCACATACTTGAG ACTGTTTTGA ATTTGGGGGA TGGCTAAAAC 3030 CATCATAGTA CAGGTAAGGTGAGGGAATAG TAAGTGGTGA GAACTACTCA GGGAATGAAG 3090 GTGTCAGAAT AATAAGAGGTGCTACTGACT TTCTCAGCCT CTGAATATGA ACGGTGAGCA 3150 TTGTGGCTGT CAGCAGGAAGCAACGAAGGG AAATGTCTTT CCTTTTGCTC TTAAGTTGTG 3210 GAGAGTGCAA CAGTAGCATAGGACCCTACC CTCTGGGCCA AGTCAAAGAC ATTCTGACAT 3270 CTTAGTATTT GCATATTCTTATGTATGTGA AAGTTACAAA TTGCTTGAAA GAAAATATGC 3330 ATCTAATAAA AAACACCTTCTAAAATAAAA AAAAAAAAAA AAAAAAAAAA AAA 3383 (2) INFORMATION FOR SEQ IDNO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 352 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “88C aminoacid sequence” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asp Tyr GlnVal Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 Ser Glu ProCys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 Leu Pro ProLeu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 Met Leu ValIle Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met 50 55 60 Thr Asp IleTyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 Leu ThrVal Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 Gly AsnThr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110 PheSer Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120 125Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe 130 135140 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser 145150 155 160 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu HisTyr 165 170 175 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe TrpLys Asn 180 185 190 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val LeuPro Leu Leu 195 200 205 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys ThrLeu Leu Arg Cys 210 215 220 Arg Asn Glu Lys Lys Arg His Arg Ala Val ArgLeu Ile Phe Thr Ile 225 230 235 240 Met Ile Val Tyr Phe Leu Phe Trp AlaPro Tyr Asn Ile Val Leu Leu 245 250 255 Leu Asn Thr Phe Gln Glu Phe PheGly Leu Asn Asn Cys Ser Ser Ser 260 265 270 Asn Arg Leu Asp Gln Ala MetGln Val Thr Glu Thr Leu Gly Met Thr 275 280 285 His Cys Cys Ile Asn ProIle Ile Tyr Ala Phe Val Gly Glu Lys Phe 290 295 300 Arg Asn Tyr Leu LeuVal Phe Phe Gln Lys His Ile Ala Lys Arg Phe 305 310 315 320 Cys Lys CysCys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser 325 330 335 Ser ValTyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350 (2)INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1915 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:CDS (B) LOCATION: 362..1426 (ix) FEATURE: (A) NAME/KEY: misc_feature (D)OTHER INFORMATION: /= “88-2B polynucleotide and amino acid sequences”(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATAATAATGA TTATTATATT GTTATCATTATCTAGCCTGT TTTTTCCTGT TTTGTATTTC 60 TTCCTTTAAA TGCTTTCAGA AATCTGTATCCCCATTCTTC ACCACCACCC CACAACATTT 120 CTGCTTCTTT TCCCATGCCG GGTCATGCTAACTTTGAAAG CTTCAGCTCT TTCCTTCCTC 180 AATCCTTTTC CTGGCACCTC TGATATGCCTTTTGAAATTC ATGTTAAAGA ATCCCTAGGC 240 TGCTATCACA TGTGGCATCT TTGTTGAGTACATGAATAAA TCAACTGGTG TGTTTTACGA 300 AGGATGATTA TGCTTCATTG TGGGATTGTATTTTTCTTCT TCTATCACAG GGAGAAGTGA 360 A ATG ACA ACC TCA CTA GAT ACA GTTGAG ACC TTT GGT ACC ACA TCC 406 Met Thr Thr Ser Leu Asp Thr Val Glu ThrPhe Gly Thr Thr Ser 1 5 10 15 TAC TAT GAT GAC GTG GGC CTG CTC TGT GAAAAA GCT GAT ACC AGA GCA 454 Tyr Tyr Asp Asp Val Gly Leu Leu Cys Glu LysAla Asp Thr Arg Ala 20 25 30 CTG ATG GCC CAG TTT GTG CCC CCG CTG TAC TCCCTG GTG TTC ACT GTG 502 Leu Met Ala Gln Phe Val Pro Pro Leu Tyr Ser LeuVal Phe Thr Val 35 40 45 GGC CTC TTG GGC AAT GTG GTG GTG GTG ATG ATC CTCATA AAA TAC AGG 550 Gly Leu Leu Gly Asn Val Val Val Val Met Ile Leu IleLys Tyr Arg 50 55 60 AGG CTC CGA ATT ATG ACC AAC ATC TAC CTG CTC AAC CTGGCC ATT TCG 598 Arg Leu Arg Ile Met Thr Asn Ile Tyr Leu Leu Asn Leu AlaIle Ser 65 70 75 GAC CTG CTC TTC CTC GTC ACC CTT CCA TTC TGG ATC CAC TATGTC AGG 646 Asp Leu Leu Phe Leu Val Thr Leu Pro Phe Trp Ile His Tyr ValArg 80 85 90 95 GGG CAT AAC TGG GTT TTT GGC CAT GGC ATG TGT AAG CTC CTCTCA GGG 694 Gly His Asn Trp Val Phe Gly His Gly Met Cys Lys Leu Leu SerGly 100 105 110 TTT TAT CAC ACA GGC TTG TAC AGC GAG ATC TTT TTC ATA ATCCTG CTG 742 Phe Tyr His Thr Gly Leu Tyr Ser Glu Ile Phe Phe Ile Ile LeuLeu 115 120 125 ACA ATC GAC AGG TAC CTG GCC ATT GTC CAT GCT GTG TTT GCCCTT CGA 790 Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe Ala LeuArg 130 135 140 GCC CGG ACT GTC ACT TTT GGT GTC ATC ACC AGC ATC GTC ACCTGG GGC 838 Ala Arg Thr Val Thr Phe Gly Val Ile Thr Ser Ile Val Thr TrpGly 145 150 155 CTG GCA GTG CTA GCA GCT CTT CCT GAA TTT ATC TTC TAT GAGACT GAA 886 Leu Ala Val Leu Ala Ala Leu Pro Glu Phe Ile Phe Tyr Glu ThrGlu 160 165 170 175 GAG TTG TTT GAA GAG ACT CTT TGC AGT GCT CTT TAC CCAGAG GAT ACA 934 Glu Leu Phe Glu Glu Thr Leu Cys Ser Ala Leu Tyr Pro GluAsp Thr 180 185 190 GTA TAT AGC TGG AGG CAT TTC CAC ACT CTG AGA ATG ACCATC TTC TGT 982 Val Tyr Ser Trp Arg His Phe His Thr Leu Arg Met Thr IlePhe Cys 195 200 205 CTC GTT CTC CCT CTG CTC GTT ATG GCC ATC TGC TAC ACAGGA ATC ATC 1030 Leu Val Leu Pro Leu Leu Val Met Ala Ile Cys Tyr Thr GlyIle Ile 210 215 220 AAA ACG CTG CTG AGG TGC CCC AGT AAA AAA AAG TAC AAGGCC ATC CGG 1078 Lys Thr Leu Leu Arg Cys Pro Ser Lys Lys Lys Tyr Lys AlaIle Arg 225 230 235 CTC ATT TTT GTC ATC ATG GCG GTG TTT TTC ATT TTC TGGACA CCC TAC 1126 Leu Ile Phe Val Ile Met Ala Val Phe Phe Ile Phe Trp ThrPro Tyr 240 245 250 255 AAT GTG GCT ATC CTT CTC TCT TCC TAT CAA TCC ATCTTA TTT GGA AAT 1174 Asn Val Ala Ile Leu Leu Ser Ser Tyr Gln Ser Ile LeuPhe Gly Asn 260 265 270 GAC TGT GAG CGG AGC AAG CAT CTG GAC CTG GTC ATGCTG GTG ACA GAG 1222 Asp Cys Glu Arg Ser Lys His Leu Asp Leu Val Met LeuVal Thr Glu 275 280 285 GTG ATC GCC TAC TCC CAC TGC TGC ATG AAC CCG GTGATC TAC GCC TTT 1270 Val Ile Ala Tyr Ser His Cys Cys Met Asn Pro Val IleTyr Ala Phe 290 295 300 GTT GGA GAG AGG TTC CGG AAG TAC CTG CGC CAC TTCTTC CAC AGG CAC 1318 Val Gly Glu Arg Phe Arg Lys Tyr Leu Arg His Phe PheHis Arg His 305 310 315 TTG CTC ATG CAC CTG GGC AGA TAC ATC CCA TTC CTTCCT AGT GAG AAG 1366 Leu Leu Met His Leu Gly Arg Tyr Ile Pro Phe Leu ProSer Glu Lys 320 325 330 335 CTG GAA AGA ACC AGC TCT GTC TCT CCA TCC ACAGCA GAG CCG GAA CTC 1414 Leu Glu Arg Thr Ser Ser Val Ser Pro Ser Thr AlaGlu Pro Glu Leu 340 345 350 TCT ATT GTG TTT TAGGTCAGAT GCAGAAAATTGCCTAAAGAG GAAGGACCAA 1466 Ser Ile Val Phe 355 GGAGATGAAG CAAACACATTAAGCCTTCCA CACTCACCTC TAAAACAGTC CTTCAAACTT 1526 CCAGTGCAAC ACTGAAGCTCTTGAAGACAC TGAAATATAC ACACAGCAGT AGCAGTAGAT 1586 GCATGTACCC TAAGGTCATTACCACAGGCC AGGGGCTGGG CAGCGTACTC ATCATCAACC 1646 CTAAAAAGCA GAGCTTTGCTTCTCTCTCTA AAATGAGTTA CCTACATTTT AATGCACCTG 1706 AATGTTAGAT AGTTACTATATGCCGCTACA AAAAGGTAAA ACTTTTTATA TTTTATACAT 1766 TAACTTCAGC CAGCTATTGATATAAATAAA ACATTTTCAC ACAATACAAT AAGTTAACTA 1826 TTTTATTTTC TAATGTGCCTAGTTCTTTCC CTGCTTAATG AAAAGCTTGT TTTTTCAGTG 1886 TGAATAAATA ATCGTAAGCAACAAAAAAA 1915 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 355 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88-2B amino acid sequence” (xi)SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Thr Thr Ser Leu Asp Thr Val GluThr Phe Gly Thr Thr Ser Tyr 1 5 10 15 Tyr Asp Asp Val Gly Leu Leu CysGlu Lys Ala Asp Thr Arg Ala Leu 20 25 30 Met Ala Gln Phe Val Pro Pro LeuTyr Ser Leu Val Phe Thr Val Gly 35 40 45 Leu Leu Gly Asn Val Val Val ValMet Ile Leu Ile Lys Tyr Arg Arg 50 55 60 Leu Arg Ile Met Thr Asn Ile TyrLeu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80 Leu Leu Phe Leu Val Thr LeuPro Phe Trp Ile His Tyr Val Arg Gly 85 90 95 His Asn Trp Val Phe Gly HisGly Met Cys Lys Leu Leu Ser Gly Phe 100 105 110 Tyr His Thr Gly Leu TyrSer Glu Ile Phe Phe Ile Ile Leu Leu Thr 115 120 125 Ile Asp Arg Tyr LeuAla Ile Val His Ala Val Phe Ala Leu Arg Ala 130 135 140 Arg Thr Val ThrPhe Gly Val Ile Thr Ser Ile Val Thr Trp Gly Leu 145 150 155 160 Ala ValLeu Ala Ala Leu Pro Glu Phe Ile Phe Tyr Glu Thr Glu Glu 165 170 175 LeuPhe Glu Glu Thr Leu Cys Ser Ala Leu Tyr Pro Glu Asp Thr Val 180 185 190Tyr Ser Trp Arg His Phe His Thr Leu Arg Met Thr Ile Phe Cys Leu 195 200205 Val Leu Pro Leu Leu Val Met Ala Ile Cys Tyr Thr Gly Ile Ile Lys 210215 220 Thr Leu Leu Arg Cys Pro Ser Lys Lys Lys Tyr Lys Ala Ile Arg Leu225 230 235 240 Ile Phe Val Ile Met Ala Val Phe Phe Ile Phe Trp Thr ProTyr Asn 245 250 255 Val Ala Ile Leu Leu Ser Ser Tyr Gln Ser Ile Leu PheGly Asn Asp 260 265 270 Cys Glu Arg Ser Lys His Leu Asp Leu Val Met LeuVal Thr Glu Val 275 280 285 Ile Ala Tyr Ser His Cys Cys Met Asn Pro ValIle Tyr Ala Phe Val 290 295 300 Gly Glu Arg Phe Arg Lys Tyr Leu Arg HisPhe Phe His Arg His Leu 305 310 315 320 Leu Met His Leu Gly Arg Tyr IlePro Phe Leu Pro Ser Glu Lys Leu 325 330 335 Glu Arg Thr Ser Ser Val SerPro Ser Thr Ala Glu Pro Glu Leu Ser 340 345 350 Ile Val Phe 355 (2)INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “V28degf2” (xi) SEQUENCEDESCRIPTION: SEQ ID NO:5: GACGGATCCA TYGAYAGRTA CCTGGCYATY GTCC 34 (2)INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “V28degr2” (xi) SEQUENCEDESCRIPTION: SEQ ID NO:6: GCTAAGCTTT TRTAGGGDGT CCAYAAGAGY AA 32 (2)INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88c-r4” (xi) SEQUENCEDESCRIPTION: SEQ ID NO:7: GATAAGCCTC ACAGCCCTGT G 21 (2) INFORMATION FORSEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: misc_feature (D) OTHERINFORMATION: /= “88c-rlb” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GCTAAGCTTG ATGACTATCT TTAATGTC 28 (2) INFORMATION FOR SEQ ID NO:9: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (ix) FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /=“88-2B-3” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCCTCTAGAC TAAAACACAATAGAGAG 27 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “88-2B-5”(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GCTAAGCTTA TCACAGGGAGAAGTGAAATG 30 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “88-2B-f1”(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AGTGCTAGCA GCTCTTCCTG 20 (2)INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “88-2B-r1” (xi) SEQUENCEDESCRIPTION: SEQ ID NO:12: CAGCAGCGTT TTGATGATTC 20 (2) INFORMATION FORSEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: misc_feature (D)OTHER INFORMATION: /= “88c-f1” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:TGTGTTTGCT TTAAAAGCC 19 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “88C-r3”(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TAAGCCTCAC AGCCCTG 17 (2)INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY:misc_feature (D) OTHER INFORMATION: /= “CCCKR1(2)-5 Primer” (xi)SEQUENCE DESCRIPTION: SEQ ID NO:15: CGTAAGCTTA GAGAAGCCGG GATGGGAA 28(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iv) ANTI-SENSE: YES (ix)FEATURE: (A) NAME/KEY: misc_feature (D) OTHER INFORMATION: /= “CCCKR-3Primer” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCCTCTAGAG TCAGAGACCAGCAGA 25

We claim:
 1. A purified and isolated polypeptide comprising thechemokine receptor 88C. amino acid sequence set forth in SEQ ID NO:2.